According to the Centers for Disease Control and Prevention, antibiotic-resistant infections are already linked to 23,000 deaths and 2 million illnesses in the United States each year. Many of the mortality cases are associated with life-threatening complications, especially sepsis. Estimates of the economic impact vary, but have ranged as high as $20 billion in excess direct healthcare costs, and as much as $35 billion in lost productivity from hospitalizations and sick days. Unfortunately, the problem is worsening because of an alarming increase in antibiotic-resistant bacteria in recent years and the void in the development and discovery of new antibiotics by pharmaceutical companies over the last three decades. As a consequence, development of novel therapies to effectively combat drug-resistant bacteria is not only of scientific and medical importance, but a national priority. We have developed a series of rationally engineered cationic antimicrobial peptides (eCAPs) using different amino acids computationally arranged to achieve in vitro inactivation of diverse drug- resistant bacterial strains. One of the lead compounds, WLBU2 (made only of Arg, Val, and Trp), has demonstrated potent bactericidal activity against diverse difficult-to-treat drug resistant pathogens that have developed resistance to other membrane-active compounds, such as the natural antimicrobial peptide (AMP) LL37 and colistin, an antibiotic of last resort. In addition, we have demonstrated a substantially lower tendency for bacteria to develop resistance to WLBU2 compared to standard antibiotic agents and natural AMPs. Importantly, WLBU2 demonstrates in vivo efficacy in a murine model of P. aeruginosa sepsis when the bacteria were systemically administered. Because of an initially narrow therapeutic index (TI ? 5), we are continuously optimizing eCAP structure using a systematic iterative design approach to lower host toxicity and enhance potency and stability. Preliminary data indicate that some of these Trp-based eCAPs (collectively referred to as W2eCAPs) have already demonstrated a higher TI. Based on the exciting results from these exploratory studies, the use of W2eCAPs to overcome bacterial resistance is an appealing concept. Hence, we hypothesize that W2eCAPs will display enhanced bactericidal activities against DR bacteria as well as negligible host toxicity and, therefore, can be used as an effective therapy to treat pneumonia-induced sepsis. Due to the unique lung microenvironment, many questions remain to be answered before the clinical ideal of using W2eCAPs can be realized. Thus, the primary purpose of this proposal is to understand: 1) how W2eCAPs exert their antimicrobial activities against major respiratory drug resistant pathogens in conditions associated with the lung microenvironment; 2) the molecular mechanisms used by W2eCAPs to kill bacteria; (3) the optimal treatment regimens (systemic compared to airway delivery) to select the W2eCAP with the highest TI; and (4) the pharmacokinetic properties of the selected W2eCAPs.